In this specification polyunsaturated fatty acids are identified according to the system wherein the omega- or n-number denominates the position of the first double bond when counting from the terminal methyl group, e.g. in an omega-3 or n-3 fatty acid, the first double bond occurs at the third carbon carbon-bond from the terminal methyl group of the acid. Further, when a fatty acid is identified, for instance, as C18:3, this refers to a fatty acid having 18 carbon atoms in the chain and three double bonds.
Commercially important polyunsaturated fatty acids are EPA (eicosapentaenoic acid, C20:5) DHA (docosahexaenoic acid, C22:6) and AA (arachidonic acid, C20:4). The full nomenclature of these acids according to the IUPAC system is:
EPA PA1 DHA PA1 AA PA1 (a) subjecting the oil composition to a transesterification reaction with a C.sub.1 -C.sub.6 alcohol under substantially anhydrous conditions and in the presence of a lipase active to preferentially catalyze the transesterification of the saturated and monounsaturated fatty acids, the amount of said C.sub.1 -C.sub.6 alcohol present in the reaction being not more than 15 molar equivalents based on the triglycerides present; and thereafter PA1 (b) separating a residual fraction enriched in glycerides of polyunsaturated fatty acids from a fraction containing saturated and monounsaturated fatty acid esters produced by said lipase-catalyzed transesterification reaction. PA1 (a) subjecting the oil composition to a transesterification reaction with a C.sub.1 -C.sub.6 alcohol under substantially anhydrous conditions and in the presence of a lipase active to preferentially catalyze the transesterification of the saturated and monounsaturated fatty acids, the amount of said C.sub.1 -C.sub.6 alcohol present in the reaction being not more than 15 molar equivalents based on the triglycerides present; and thereafter PA1 (b) subjecting the product obtained in step (a) to one or more molecular distillations to recover a fraction enriched in glycerides of polyunsaturated fatty acids and from which environmental pollutants have been preferentially removed. PA1 (i) the absence of any solvent leads to a considerable reduction in bulkiness, which effect is enhanced by the ability to use only stoichiometric concentrations of alcohol, PA1 (ii) the transesterification reaction can be conducted under mild e.g. room temperature conditions, minimizing side reactions and not requiring high energy inputs, PA1 (iii) the recovery of EPA and DHA is very high, and the recovered product is essentially freed of contamination by environmental pollutants, and PA1 (iv) the substantially anhydrous reaction conditions used means that there is minimum hydrolysis, whereby molecular distillation of the glyceride fraction from the transesterification reaction gives a good separation of desired polyunsaturated fatty acids from unwanted saturated and monounsaturated fatty acids. PA1 (c) transesterifying the glyceride fraction with a lower alcohol, e.g. ethanol, using either chemical catalysis e.g. an alkaline environment containing amounts of a base such as sodium or potassium ethoxide sufficient only to catalyze the transesterification, or enzymatic catalysis e.g. using Candida antarctica lipase under substantially anhydrous conditions; PA1 (d) heating the resulting alkyl ester product with an excess of urea in an alkanol to a temperature of from 55.degree. to 99.degree. C.; PA1 (e) cooling the product of step (d), e.g. to about 0.degree.-25.degree. C. to precipitate urea fatty acid alkyl ester adduct and thereafter separating off said adduct to leave a solution mainly containing n-3 fatty acid esters; PA1 (f) separating from the solution remaining from step (e) the n-3 fatty acid alkyl esters; and PA1 (g) removing any solvent from the mixture obtained in step (f).
cis-5,8,11,14,17-eicosapentaenoic acid
cis-4,7,10,13,16,19-docosahexaenoic acid
cis-5,8,11,14-eicosatetraenoic acid
As is well known, EPA and DHA are proving increasingly valuable in the pharmaceutical and food supplement industries in particular. These acids are found in relatively high concentrations in certain marine oils, but for many purposes it is necessary that the marine oils should be refined in order to increase the content of EPA and/or DHA to suitable levels, or to reduce the concentrations of, or even eliminate, certain other substances which occur naturally in the raw oil. For pharmaceutical and food purposes, for instance, it is necessary to substantially completely eliminate all the pesticide residues which commonly occur in marine oils, even those derived from fish caught in sea areas quite remote from intensively cultivated land areas.
EPA and DHA must exhibit an all-cis (Z--Z) configuration corresponding to their naturally occurring state if they are to exhibit biological activity without toxicity. However, these acids are extremely fragile when they are heated and they very readily undergo fast oligomerization, isomerization and peroxidation reactions. Accordingly, it is extremely difficult to purify marine oil compositions containing EPA and DHA without risking loss of these desired acids in their useful form.
EPA and DHA occur in marine oils predominantly as their triglycerides. Up until now, most practical refining processes start either by esterifying the oil with a low molecular weight alcohol (normally ethanol) or by hydrolysing the oil to form free acids or their salts, whereafter fractionation of the oil to recover the desired product is initiated.
However, because of the complexity of marine raw materials, polyunsaturated fatty acid derivatives in highly purified form are not easily prepared by any single fractionation technique. Usually a combination of techniques is therefore used, the particular combination chosen depending on the composition of the raw material and the concentration and other quality criteria that are desired for the product. Urea complexation is one fractionation technique which is commonly employed in processes for recovering high content EPA and/or DRA compositions.
Urea has the useful property of forming solid complexes with straight-chain organic compounds. When a marine oil composition containing fatty acids or esters is added to a solution of urea, a crystalline complex is formed with the more saturated fraction of the acids. The crystals can be removed, leaving a raffinate of more unsaturated fatty acids or fatty acid esters.
Urea complexation has been used with both free fatty acids, and with methyl or ethyl esters of the fatty acids. The process can be made continuous by using heat exchangers with a scraped surface as reactors for urea occlusion formation. When fractionating esters, it seems to be the normal procedure first to react the oil with alcohol and/or alcohol/water and then to isolate the esters/free fatty acids before urea complexation. However, a combined in situ esterification and urea fractionation may also be performed as described in EP-A-0255824.
When such a process is used in combination with, for instance, two or more steps of molecular distillation, it is possible to produce a refined product containing 85% by weight or higher of n-3 polyunsaturated fatty acids, predominantly EPA and DHA from a raw marine oil. However, the total recovery of the refined product is undesirably low. In a typical commercial operation using such conventional fractionation processes, one might expect to recover only about 60-80 tons of an 85% n-3 fatty acid concentrate from 1000 tons of raw marine oil, i.e. a recovery rate of only 6-8%. This poor yield means not only that such refining processes are very expensive but also that they require bulky, complex equipment.
The lipophilic character of many environmental pollutants (examples: pesticides and polychlorinated biphenyls) results in an accumulation of these compounds in marine lipids. Unfortunately, urea does not form complexes with many such pollutants, and as a result a concentrate from urea complexation will contain increased, and for many purposes unacceptably high, levels of pesticides and other environmental pollution products as compared with the original marine oil. Consequently, current refining processes based on urea complexation for making refined fish oils for human consumption have to include complex and expensive purification procedures to reduce pollutant levels to acceptable values.
The present invention aims to provide an improved process for increasing the polyunsaturated fatty acid content of oil compositions, and particularly a process which is well adapted to a commercial process for recovering EPA and/or DHA from fish oil in enhanced yield.
As is known, lipases are well suited for use as catalysts in processes involving highly labile n-3 polyunsaturated fatty acids, such as EPA and DHA, occurring in marine oil. This is due to their ability to act at low temperatures, their neutral pH and their mildness in action, which helps keep to a minimum undesired side reactions such as cis-trans isomerizations, double-bond migrations, polymerizations, oxidations, etc. Thus, the utilization of lipases for the hydrolysis of fatty acids in marine oil is already well documented.
For instance, Lie and Lambertsen in Comp. Biochem. Physiol. 80B No. 3, pages 447-450, 1985, reported that intestinal lipase obtained from cod hydrolysed preferentially the polyunsaturated fatty acids 18:4, 20:5 and 22:6 present as triglycerides in capelin oil. They reported that this specificity was particularly prominent for the acid 20:5, i.e. EPA.
On the other hand, Lie and Lambertsen also found that the C.sub.14 -C.sub.18 saturated and monounsaturated fatty acids were preferentially hydrolysed as triglycerides from capelin oil by Candida cylinracea lipase, whereas the long-chain monoenes, C20:1 and C22:1 and particularly the polyunsaturated fatty acids C18:4, and, to a lesser extent EPA, DHA were resistant to hydrolysis (Lie and Lambertsen, Fette, Seifen, Anstrichmittel, 88, 365, 1986).
A Japanese patent specification No. 59-14793--Noguchi et al, describes a method based on similar discrimination by lipases between saturated and unsaturated acids for is preparing concentrates of highly polyunsaturated fatty acids. Ethyl esters from miscellaneous marine oils such as sardine and mackerel oil were hydrolysed with various lipases (Candida cylinracea, Aspergillus rhizopus and Mucor miehei). Selective hydrolysis afforded ethyl ester concentrates of up to 25% EPA and 17% DHA after separation of the hydrolysed fatty acids.
Another Japanese patent, 172691--Nippon Oil and Fats, 1990 describes a method based on the hydrolysis of marine oil with Candida sp. lipase. EPA or its ester was obtained from a free fatty acid component and DHA or its ester from the residual glyceride component. The continuation of the process involved separation of fatty acid and glyceride components, esterification with lower alkyl, concentration of polyunsaturated fatty acids using urea complexion and further purification by molecular distillation, super-critical CO.sub.2 fluid extraction or liquid chromatography. Takagi (Am. Oil Chem. Soc. 66, 488, 1988) has described a method based on the reverse process for separating EPA and DHA using an immobilized Mucor miehei lipase.
Polyunsaturated fatty acid concentrates obtained from Japanese sardine oil by the urea adduct method were esterified with methanol in n-hexane medium at room temperature. The lipase discriminated between EPA and DHA and selective esterification afforded an EPA enriched methyl ester concentrate of 5% EPA and 6% DHA as well as a DHA enriched free fatty acid concentrate of 52% DHA and 12% EPA in the ratio of 59 to 41, respectively.
Yamane and co-workers have very recently used several lipases to selectively hydrolyse cod liver oil and refined sardine oil (Agric. Biol. Chem. 54, 1459, 1990). The best results were obtained for the non-regiospecific Candida cylindracea and 1,3-specific Aspergillus niger lipases, but none of the lipases were able to raise the EPA content of the glyceride products significantly.
It is apparent from this prior work on the use of lipases to hydrolyse esters of marine oil fatty acids that different lipases behave quite differently, and also that a quite marked selectivity between one fatty acid and another, or between one type of fatty acid and another, is often exhibited by the lipases.
This substrate selectivity of certain lipases is taken advantage of by Zuyi and Ward who have described a lipase-catalysed alcoholysis of cod liver oil to prepare an n-3 polyunsaturated fatty acid-enriched fraction (Zuyi and Ward, "Lipase-catalyzed alcoholysis to concentrate the n-3 polyunsaturated fatty acid of cod liver oil", Enzyme Microb. Technol., 1993, 15, July, 601-606). The authors studied nine lipases and they found that Pseudomonas sp. lipase (CES from Amano International Enzyme Co) alcoholized fish oil at a higher rate than the other lipases and produced monoglycerides significantly enriched in EPA and DHA. In this prior process, the alcohol (ethanol and isopropanol were used) was employed both as a reactant and as a solvent for the reaction.
Zuyi and Ward studied the effect of water concentration in the reaction medium and they found that the alcoholysis (with isopropanol) by lipase CES increased with water content in the range 0-7.5% v/v. The data presented shows that a water content of 5% v/v was optimal, and that even with a water content of 2.5% v/v over 40% of the original triglycerides present in the oil remained unreacted after 12 hours (it is understood that these water contents refer to water added to the reaction system and take no account of the small amounts of water inevitably present in the fish oil and the "dry" enzyme). Concomitantly, there was very considerable hydrolysis of triglyceride to free fatty acid (typically more than 30%), for instance at a water content of 5% v/v there was 18.9% hydrolysis (compared with only 15.5% alcoholysis).
Although the Zuyi and Ward process is of scientific interest it does not, unfortunately, provide an improved process for preparing purified EPA/DHA compositions commercially. In particular, the inevitable presence in the lipase-catalysed product of significant quantities of free fatty acid make it difficult subsequently to purify the desired n-3 polyunsaturated fatty acids. For example, due to low volatility the free fatty acids cannot be separated from the glycerides by conventional molecular distillation techniques. Furthermore, since the esters and the free fatty acids have substantially different polarities, whilst the mono- and di-glycerides are of intermediate polarities, they cannot be separated from the glycerides using extraction. As demonstrated below, the present invention in contrast advantageously may use molecular distillation techniques not only to separate polyunsaturated fatty acid glycerides from saturated and monounsaturated fatty acid esters but also to simultaneously effect removal of environmental pollutants such as pesticides and polychlorinated biphenyls from the desired polyunsaturated fatty acid glyceride fraction.